4,5-Dihydroxyanthraquinone-2-carboxylic acid (Rhein) has been shown to have various physiological and pharmacologicalproperties including anticancer activity and modulatory effects on bioenergetics. In this study, we explored the impact ofrhein on protein profiling of undifferentiated (UC) and differentiated (DC) SH-SY5Y cells. Besides that, the cellularmorphology and expression of differentiation markers were investigated to determine the effect of rhein on retinoic acidinducedneuronal cell differentiation. Using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry we evaluated the changes in the proteome of both UC and DC SH-SY5Y cellsafter 24 h treatment with rhein. Validation of selected differentially expressed proteins and the assessment of neuronaldifferentiation markers were performed by western blotting. Proteomic analysis revealed significant changes in theabundance of 15 proteins linked to specific cellular processes such as cytoskeleton structure and regulation, mitochondrialfunction, energy metabolism, protein synthesis and neuronal plasticity. We also observed that the addition of rhein to thecultured cells during differentiation resulted in a significantly reduced neurite outgrowth and decreased expression ofneuronal markers. These results indicate that rhein may strongly interfere with the differentiation process of SH-SY5Yneuroblastoma cells and is capable of inducing marked proteomic changes in these cells.